Like many transmembrane proteins, determination of the structure of P-gp by X-ray crystallography has proven elusive despite much effort. This stems from the difficulty of forming sufficient-quality crystals that maintain the native physiochemical environments for the different parts of the protein. Thus, in lieu of direct experimental determination, we are striving to integrate all available (often indirect) experimental data with physiochemically-based mathematical methods to produce one or more physically realistic models of the structure. Fortunately, over three decades of study has provided a wealth of information about P-gp from which we can gleam structural information. On a broad scale, it is known that the protein is composed of two homologous domains, each with a six-segment transmembrane component and a nucleotide-binding component. To date, the best two sources of structural information about P-gp are an X-ray crystal structure of the homologous bacterial protein Sav1866, and low-resolution cryo-electron micrographs of human P-gp. In addition, the corrected X-ray structure of the bacterial lipid flippase MsbA is expected soon, which is even more closely related to P-gp than is Sav1866. Thus, one major focus of our work is to develop a homology model of human P-gp using the crystal structures of the bacterial proteins as templates. While another group has recently published such a model, it was only based on a simple alignment of the P-gp and Sav1866 sequences, and unfortunately, this is not well defined for the transmembrane segments. Rather, our efforts go deeper into examining the patterns of residue conservation within the family of closely related MDR proteins and the superfamily of ABC transporters. This information helps predict which residues are exposed to the core and headgroup layers of the membrane, which residues line the pore, and which are at the interfaces of the two transmembrane domains. We are currently in the process of developing a grand sequence alignment of homologous families and the superfamily. The results of this will also enable the determination of patterns of correlated mutations, which help identify groups of residues that are proximal in the 3-dimensional structure of the protein. Finally, we will examine the resultant models for consistency with all the experimental data, such as the effects of site-directed mutagenesis, naturally occurring polymorphisms, and cross-linking data. Where the model based on the Sav1866 template fails to explain the experimental results, we will search for alternate conformations that bring it into compliance. The other major focus is to develop models from the density maps obtained from electron microscopy. This has the advantage that the structural data is directly from human P-gp, the target protein, and not from a bacterial homolog, which likely differs in structure. This includes the fact that the two transmembrane domains of human P-gp are different in sequence, and thus are asymmetrical around the approximate two-fold axis of the pore (as observed in the micrographs), while the bacterial homologs only contain one domain, and thus form perfectly symmetrical homodimers in the membrane. Having obtained coordinates of generic helices fitted to the transmembrane electron density, we have nearly completed computer software to determine the alignment of the amino acids sequences onto the backbone structure of the protein. This amounts to a typical Protein Threading procedure on a grand computational scale. The program builds every possible combination of amino acid segment to transmembrane helix and scores it according to a series of physiochemical and experimentally-determined criteria. These include the proper environment for each type of amino acid residue, the clustering of conserved residues, and experimental cross-linking distance constraints, One difficulty that had to be surmounted was the relative enormous number of permutations that had to be scanned. This was overcome by developing clever algorithms to reduce the required amount of computations to within the practical scope of present-day technological capabilities. As the models develop, we will be working closely with other groups within the Lab of Cell Biology, especially Dr.s Di Xia's, Suresh Ambudkar's, and Michael Gottesman's sections, to undertake experimental tests, X-ray structure determination, and inhibitor development.